HSP27 phosphorylation modulates TRAIL-induced activation of Src-Akt/ERK signaling through interaction with β-arrestin2

Heat shock protein 27 (HSP27) regulates critical cellular functions such as development, differentiation, cell growth and apoptosis. A variety of stimuli induce the phosphorylation of HSP27, which affects its cellular functions. However, most previous studies focused on the role of HSP27 protein itself in apoptosis, the particular role of its phosphorylation state in signaling transduction remains largely unclear. In the present study, we reported that HSP27 phosphorylation modulated TRAIL-triggered pro-survival signaling transduction. In HeLa cells, suppression of HSP27 phosphorylation by specific inhibitor KRIBB3 or MAPKAPK2 (MK2) knockdown and by overexpression of non-phosphorylatable HSP27(3A) mutant demonstrated that hindered HSP27 phosphorylation enhanced the TRAIL-induced apoptosis. In addition, reduced HSP27 phosphorylation by KRIBB3 treatment or MK2 knockdown attenuated the TRAIL-induced activation of Akt and ERK survival signaling through suppressing the phosphorylation of Src. By overexpression of HSP27(15A) or HSP27(78/82A) phosphorylation mutant, we further showed that phosphorylation of HSP27 at serine 78/82 residues was essential to TRAIL-triggered Src-Akt/ERK signaling transduction. Co-immunoprecipitation and confocal microscopy showed that HSP27 interacted with Src and scaffolding protein β-arrestin2 in response of TRAIL stimulation and suppression of HSP27 phosphorylation apparently disrupted the TRAIL-induced interaction of HSP27 and Src or interaction of HSP27 and β-arrestin2. We further demonstrated that β-arrestin2 mediated HSP27 action on TRAIL-induced Src activation, which was achieved by recruiting signaling complex of HSP27/β-arrestin2/Src in response to TRAIL. Taken together, our study revealed that HSP27 phosphorylation modulates TRAIL-triggered activation of Src-Akt/ERK pro-survival signaling via interacting with β-arrestin2 in HeLa cells.     

α-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases

α-Tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with α-tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2 × 10⁶ cells. A saturating dose of TS (40 μmol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 μmol/l), and much more than Trolox (40 μmol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of α-tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that α-tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination

Cytochrome c is a highly conserved protein, with 20 residues identical in all eukaryotic cytochromes c. Gly-41 is one of these invariant residues, and is the position of the only reported naturally occurring mutation in cytochrome c (human G41S). The basis, if any, for the conservation of Gly-41 is unknown. The mutation of Gly-41 to Ser enhances the apoptotic activity of cytochrome c without altering its role in mitochondrial electron transport. Here we have studied additional residue 41 variants and determined their effects on cytochrome c functions and conformation. A G41T mutation decreased the ability of cytochrome c to induce caspase activation and decreased the redox potential, whereas a G41A mutation had no impact on caspase induction but the redox potential increased. All residue 41 variants decreased the pK _a of a structural transition of oxidized cytochrome c to the alkaline conformation, and this correlated with a destabilization of the interaction of Met-80 with the heme iron(III) at physiological pH. In reduced cytochrome c the G41T and G41S mutations had distinct effects on a network of hydrogen bonds involving Met-80, and in G41T the conformational mobility of two Ω-loops was altered. These results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.     

Mg(2+) modulates voltage-dependent activation in ether-à-go-go potassium channels by binding between transmembrane segments S2 and S3

Extracellular Mg(2+) directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg(2+) presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg(2+)-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K(+) channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg(2+) on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg(2+)-sensitivity and mimicked the slowing of activation kinetics caused by Mg(2+) binding to the wild-type channel. These results suggest that Mg(2+) modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg(2+), indicating that D284 and D319 do not mediate the slowing of activation caused by Mg(2+) binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg(2+)-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg(2+) sensitivity but is in the vicinity of the binding site. We conclude that Mg(2+) binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K(+) channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.     

Dietary sodium modulates the interaction between efferent and afferent renal nerve activity by altering activation of α2-adrenoceptors on renal sensory nerves

Activation of efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA), which then reflexively decreases ERSNA via activation of the renorenal reflexes to maintain low ERSNA. The ERSNA-ARNA interaction is mediated by norepinephrine (NE) that increases and decreases ARNA by activation of renal α(1)-and α(2)-adrenoceptors (AR), respectively. The ERSNA-induced increases in ARNA are suppressed during a low-sodium (2,470 ± 770% s) and enhanced during a high-sodium diet (5,670 ± 1,260% s). We examined the role of α(2)-AR in modulating the responsiveness of renal sensory nerves during low- and high-sodium diets. Immunohistochemical analysis suggested the presence of α(2A)-AR and α(2C)-AR subtypes on renal sensory nerves. During the low-sodium diet, renal pelvic administration of the α(2)-AR antagonist rauwolscine or the AT1 receptor antagonist losartan alone failed to alter the ARNA responses to reflex increases in ERSNA. Likewise, renal pelvic release of substance P produced by 250 pM NE (from 8.0 ± 1.3 to 8.5 ± 1.6 pg/min) was not affected by rauwolscine or losartan alone. However, rauwolscine+losartan enhanced the ARNA responses to reflex increases in ERSNA (4,680 ± 1,240%·s), and renal pelvic release of substance P by 250 pM NE, from 8.3 ± 0.6 to 14.2 ± 0.8 pg/min. During a high-sodium diet, rauwolscine had no effect on the ARNA response to reflex increases in ERSNA or renal pelvic release of substance P produced by NE. Losartan was not examined because of low endogenous ANG II levels in renal pelvic tissue during a high-sodium diet. Increased activation of α(2)-AR contributes to the reduced interaction between ERSNA and ARNA during low-sodium intake, whereas no/minimal activation of α(2)-AR contributes to the enhanced ERSNA-ARNA interaction under conditions of high sodium intake.     

Functional interactions between the oxytocin receptor and the β2-adrenergic receptor: implications for ERK1/2 activation in human myometrial cells

The Gq-coupled oxytocin receptor (OTR) and the Gs-coupled β₂-adrenergic receptor (β₂AR) are both expressed in myometrial cells and mediate uterine contraction and relaxation, respectively. The two receptors represent important pharmacological targets as OTR antagonists and β₂AR agonists are used to control pre-term uterine contractions. Despite their physiologically antagonistic effects, both receptors activate the MAP kinases ERK1/2, which has been implicated in uterine contraction and the onset of labor. To determine the signalling pathways involved in mediating the ERK1/2 response, we assessed the effect of blockers of specific G protein-associated pathways. In human myometrial hTERT-C3 cells, inhibition of Gαi as well as inhibition of the Gαq/PKC pathway led to a reduction of both OTR- and β₂AR-mediated ERK1/2 activation. The involvement of Gαq/PKC in β₂AR-mediated ERK1/2 induction was unexpected. To test whether the emergence of this novel signalling mechanism was dependent on OTR expression in the same cell, we conducted experiments in HEK 293 cells that were transfected with the β₂AR alone or co-transfected with the OTR. Using this approach, we found that β₂AR-mediated ERK1/2 responses became sensitive to PKC inhibition only in cells co-transfected with the OTR. Inhibitor studies indicated the involvement of an atypical PKC isoform in this process. We confirmed the specific involvement of PKCζ in this pathway by assessing PKCζ translocation to the cell membrane. Consistent with our inhibitor studies, we found that β₂AR-mediated PKCζ translocation was dependent on co-expression of OTR. The present demonstration of a novel β₂AR-coupled signalling pathway that is dependent on OTR co-expression is suggestive of a molecular interaction between the two receptors.     

Lipopolysaccharide stimulates syntheses of toll-like receptor 2 and surfactant protein-A in human alveolar epithelial A549 cells through upregulating phosphorylation of MEK1 and ERK1/2 and sequential activation of NF-κB

Surfactant proteins (SPs) and toll-like receptors (TLRs) contribute to regulation of sepsis-induced acute lung injury. Lipopolysaccharide (LPS) is one of the major causes of septic shock. This study was designed to evaluate the effects of LPS on the regulation of tlr-2 and sp-a gene expression in human alveolar epithelial A549 cells and the possible mechanisms. Exposure of A549 cells to LPS increased the expressions of TLR2 and SP-A mRNA and protein in time-dependent manners. A search using a bioinformatic approach found that there are several nuclear factor kappa-B (NF-κB)-DNA-binding motifs in the promoter region of the tlr2 and sp-a genes. Immunoblotting analyses revealed that exposure to LPS time-dependently enhanced the translocation of NF-κB from the cytoplasm to nuclei. Analyses of an electrophoretic mobility shift assay further showed that LPS augmented the transactivation activity of NF-κB to its consensus oligonucleotides in A549cells. Sequentially, treatment of A549 cells with LPS increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38-mitogen-activated protein kinase (p38MAPK), and MAPK kinase-1 (MEK1). Pretreatment with PD98059, an inhibitor of ERK1/2, significantly decreased LPS-induced TLR2 and SP-A mRNA expression.     

Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization

The oxytocin receptor (OTR) and the β₂-adrenergic receptor (β₂AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β₂AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β₂AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β₂AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β₂AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β₂AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β₂AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β₂AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β₂AR in the context of a new heterodimer pair lie at the heart of the allosteric effects.     

Cation mediation of radical transfer between Trp48 and Tyr356 during O2 activation by protein R2 of Escherichia coli ribonucleotide reductase: relevance to R1-R2 radical transfer in nucleotide reduction?

A tryptophan 48 cation radical (W48(+)(*)) forms concomitantly with the Fe(2)(III/IV) cluster, X, during activation of oxygen for tyrosyl radical (Y122.) production in the R2 subunit of class I ribonucleotide reductase (RNR) from Escherichia coli. W48(+)(*) is also likely to be an intermediate in the long-range radical transfer between R2 and its partner subunit, R1, during nucleotide reduction by the RNR holoenzyme. The kinetics of decay of W48(+)(*) and formation of tyrosyl radicals during O(2) activation (in the absence of R1) in wild-type (wt) R2 and in variants with either Y122, Y356 (the residue thought to propagate the radical from W48(+)(*) into R1 during turnover), or both replaced by phenylalanine (F) have revealed that the presence of divalent cations at concentrations similar to the [Mg(2+)] employed in the standard RNR assay (15 mM) mediates a rapid radical-transfer equilibrium between W48 and Y356. Cation-mediated propagation of the radical from W48 to Y356 gives rise to a fast phase of Y. production that is essentially coincident with W48(+)(*) formation and creates an efficient pathway for decay of W48(+)(*). Possible mechanisms of this cation mediation and its potential relevance to intersubunit radical transfer during nucleotide reduction are considered.